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Techniques of Molecular Medicine > Testing the sample quality > Flashcards

Testing the sample quality Flashcards

1
Q

What is genomics used for?

A

Ancestry

Crime

Disease

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2
Q

What event revolutionised genomics?

A

Human Genome Project

2003

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3
Q

What is the most important part of a scientific experiment?

A

Obtaining an appropriate sample

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4
Q

What characterises as an appropriate sample in microarrays?

A

Good quality

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5
Q

What are ways in which the source used in genome investigations can differ?

A

Source

Type

Species

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6
Q

Different sources of genetic samples

A

Cells

Tissues

Blood

Saliva

Bacteria

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7
Q

Different types of genetic samples

A

Fresh

Cultured

Frozen

Fixed

Archived

Laser capture

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8
Q

Different species of genetic samples

A

Human

Mouse

Fish

Insect

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9
Q

What 3 things must we know about a sample in order to carry out an investigation?

A

Concentration

Integrity

Contaminants

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10
Q

What does agarose gel help us determine?

A

Whether our DNA sample is degraded or not

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11
Q

How does agarose gel tell us whether DNA is degraded or not?

A

Nucleic acid, which is negatively charged, will move towards the positive electrode and form bands

The less concentrated the DNA sample is at the electrode end, the lower quality the sample is since it is more degraded

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12
Q

What is the limitation of agarose gel?

A

Qualitative

Not tell the investigator about the quality or concentration of the sample

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13
Q

Where should a high quality DNA give a major band on the agarose gel?

A

10-20kb

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14
Q

What is nanodrop useful for?

A

Assessing the purity of DNA and RNA

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15
Q

How big are the samples measured by a nandrop?

A

1 nanol

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16
Q

Describe how a nanodrop assesses the purity of DNA or RNA

A

Measures the absorbance of the sample at 230, 260 and 280

DNA and RNA absorb wavelengths at 260

230 = salt contamination 
280 = protein contamination
17
Q

What are the results of a sample?

A

Ratio of the 260/230 and 260/280 absorbances

18
Q

What is the normal ratio of a pure DNA sample?

A

1.8-2.0

19
Q

What is the normal ratio of a pure RNA sample?

A

2.0-2.2

20
Q

How can absorbance be used to measure the sample concentration?

A

Concentration = (absorbance X (e = 50 (DNA) and 40(RNA)) / path length in cm

21
Q

What does a low ratio mean?

A

High concentration of salt/protein compared to nucleic acid

22
Q

What is the shape of the absorbance curve of a good sample?

A

Peak at 260

Does not start at origin

23
Q

What is the shape of the absorbance curve of a bad sample?

A

Peak at 230/280 depending on what the contaminant is

24
Q

If a sample is contaminated, that automatically means we cannot use it

TRUE or FALSE

A

FALSE

We need to clean the sample using a mesh to remove the contaminants

25
Q

What is the range of concentrations a nanodrop is sensitive to?

A

25 to 2000 ng/ul

26
Q

What is used to test the integrity of a sample?

A

Bioanalyzer

27
Q

What is meant by the integrity of a sample?

A

Determining whether it is degraded or not

28
Q

Describe how a bioanalyser checks for the integrity of a sample

A

Bioanalyzer contains micro-channels filled with a polymer and a fluorescent dye

Adding the sample forms an integrated electrical circuit where the charged molecules of DNA and RNA electrophoretically drive a voltage gradient

Depending on the degradation of a sample and the sybsequent peak number, the machine will generate a RIN number (RNA integrity number)

RIN makes the results easy to interpret

29
Q

What is the shape of the bioanalysis trace following RNA bioanalysis?

A

18s and 28s

Amount of RNA at these peaks is calculated by the area under the peaks

30
Q

What is the shape of a bioanalysis trace following DNA bioanalysis?

A

Simple peak

Amount of DNA at the peak is found by the height of the peak

31
Q

What is a common peak seen in all the bioanalysis traces?

A

Initial peak is the mark, which tells the scientist to start recording the trace

32
Q

What information does the bioanalyser provide?

A

Quality, integrity and concentration of the sample

Not information of contaminants

33
Q

What is the QUBIT used for?

A

Measures the concentration of nucleic acid in a sample

34
Q

Describe the process behind a QUBIT

A
  1. Sample placed into test tubes
  2. Add Qubit working solution
  3. Vortex for 2-3 seconds
  4. Incubate at room temperature
35
Q

What provides the specificity of QUBIT?

A

Fluorescent particles

Specific for the type of nucleic acid

36
Q

What informationdoes the nanodrop provide?

A

Contaminants

Concentration

37
Q

What information does the QUBIT provide?

A

Concentration

38
Q

What is the advantage of QUBIT?

A

More sensitive and accurate

Techniques of Molecular Medicine (12 decks)

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